A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Soybean 40S Ribosomal Protein S8 (GmRPS8) Interacts with 6K1 Protein and Contributes to Soybean Susceptibility to Soybean Mosaic Virus.

Soybean mosaic virus (SMV), a member of Potyvirus, is the most destructive and widespread viral disease in soybean production. Our earlier studies identified a soybean 40S ribosomal protein S8 (GmRPS8) using the 6K1 protein of SMV as the bait to screen a soybean cDNA library. The present study aims to identify the interactions between GmRPS8 and SMV and characterize the role of GmRPS8 in SMV infection in soybean. Expression analysis showed higher SMV-induced GmRPS8 expression levels in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting that GmRPS8 was involved in the response to SMV in soybean. Subcellular localization showed that GmRPS8 was localized in the nucleus. Moreover, the yeast two-hybrid (Y2H) experiments showed that GmRPS8 only interacted with 6K1 among the eleven proteins encoded by SMV. The interaction between GmRPS8 and 6K1 was further verified by a bimolecular fluorescence complementation (BiFC) assay, and the interaction was localized in the nucleus. Furthermore, knockdown of GmRPS8 by a virus-induced gene silencing (VIGS) system retarded the growth and development of soybeans and inhibited the accumulation of SMV in soybeans. Together, these results showed that GmRPS8 interacts with 6K1 and contributes to soybean susceptibility to SMV. Our findings provide new insights for understanding the role of GmRPS8 in the SMV infection cycle, which could help reveal potyviral replication mechanisms.

GmGSTU13 Is Related to the Development of Mosaic Symptoms in Soybean Plants Infected with Soybean Mosaic Virus.

The leaves of soybean cultivar ZheA8901 show various symptoms (necrosis, mosaic, and symptomless) when infected with different strains of soybean mosaic virus (SMV). Based on a proteomic analysis performed with tandem mass tags (TMT), 736 proteins were differentially expressed from soybean samples that showed asymptomatic, mosaic, and necrosis symptoms induced by SMV strains SC3, SC7, and SC15, respectively. Among these, GmGSTU13 and ascorbate peroxidase (APX) were only upregulated in mosaic and symptomless leaves, respectively. The protein level of GmGSTU13 determined by western blot analysis was consistent with TMT analysis, and quantitative reverse transcriptase PCR analysis showed that GmGSTU13 mRNA levels in mosaic plants were 5.26- and 3.75-fold higher than those in necrotic and symptomless plants, respectively. Additionally, the expression of the viral coat protein (CP) gene was increased, and serious mosaic symptoms were observed in GmGSTU13-overexpressing plants inoculated with all three SMV strains. These results showed that GmGSTU13 is associated with the development of SMV-induced mosaic symptoms in soybean and that APX is upregulated in symptomless leaves at both the transcriptional and protein levels. In APX gene-silenced soybean plants, the relative expression of the viral CP gene was 1.50, 7.59, and 1.30 times higher than in positive control plants inoculated with the three SMV strains, suggesting that the upregulation of APX may be associated with lack of symptoms in soybean infected with SMV. This work provides a useful dataset for identifying key proteins responsible for symptom development in soybean infected with different SMV strains.

Seed specifically over-expressing DGAT2A enhances oil and linoleic acid contents in soybean seeds.

Triacylglycerol (TAG), a main component of oil, is mainly biosynthesized by diacylglycerol acyltransferase (DGAT), which is critical for oil accumulation in plants. Intensive focus has been on DGAT2 functioning in unsaturated fatty acids biosynthesis. In this study, we analyzed the coding sequence (CDS) and amino acid sequence of GmDGAT2A and determined its key active sites through site-directed mutagenesis. As a consequence, H132, G201, and P152-X-I154-K155 were found to be essential active sites for GmDGAT2A. The spatial structure of the protein may bring the three active sites into close proximity, constituting an active domain. Additionally, N-terminus of GmDGAT2A was found to be an important regulator for the activity. Further, in vitro activity results uncovered GmDGAT2A was prone to utilize C18:2-CoA as the substrate. Consequently, overexpression of GmDGAT2A driven by a seed-specific promoter of Gmole1 in soybean significantly increased linoleic acid content specifically and total oil content, concomitant with accelerated elongation.

Analysis of expression characteristics of soybean leaf and root tissue-specific promoters in Arabidopsis and soybean.

The characterization of tissue-specific promoters is critical for studying the functions of genes in a given tissue/organ. To study tissue-specific promoters in soybean, we screened tissue-specific expressed genes using published soybean RNA-Seq-based transcriptome data coupled with RT-PCR analysis. We cloned the promoters of three genes, GmADR1, GmBTP1, and GmGER1, and constructed their corresponding ß-Glucuronidase (GUS) promoter-GUS reporter vectors. We generated transgenic Arabidopsis plants and examined the expression patterns of these promoters by GUS staining and RT-PCR analysis. We also transformed the promoter-GUS reporter vectors into soybean to obtain hairy roots, and examined promoter expression by GUS staining. We found a root-specific expression pattern of GmADR1 and GmBTP1 in both Arabidopsis and soybean, and the promoter of GmGER1 showed a leaf-specific pattern in transgenic Arabidopsis plants. To test the potential utility of these promoters in soybean improvement by transgenic means, we used the GmADR1 promoter to drive expression of a salt resistance gene in soybean, GmCaM4, by generating transgenic soybean plants. We found that the transgenic plants had significantly enhanced salt tolerance compared to non-transformed wild-type, suggesting that introducing endogenous promoters by transgenic means can drive the expression of functional genes in specific tissues and organs in soybean.

Dynamic analysis of pulmonary computed tomography (CT) characteristics in cured coronavirus disease 2019 (COVID-19) patients.

BACKGROUND: To retrospectively analyze the pulmonary computed tomography (CT) characteristics and dynamic changes in the lungs of cured coronavirus disease 2019 (COVID-19) patients at discharge and reexamination. METHODS: A total of 155 cured COVID-19 patients admitted to designated hospitals in Yunnan Province, China, from February 1, 2020, to March 20, 2020, were included. All patients underwent pulmonary CT at discharge and at 2 weeks after discharge (during reexamination at hospital). A retrospective analysis was performed using these two pulmonary CT scans of the cured patients to observe changes in the number, distribution, morphology, and density of lesions. RESULTS: At discharge, the lung CT images of 15 cured patients showed no obvious lesions, while those of the remaining 140 patients showed different degrees of residual lesions. Patients with moderate disease mostly had multiple pulmonary lesions, mainly in the lower lobes of both lungs. At reexamination, the lung lesions in the patients with moderate disease had significantly improved (P<0.05), and the lung lesions in the patients with severe disease had partially improved, especially in patients with multi-lobe involvement (χ 2 =3.956, P<0.05). At reexamination, the lung lesions of patients with severe disease did not show significant changes (P>0.05). CONCLUSIONS: The pulmonary CT manifestations of cured COVID-19 patients had certain characteristics and variation patterns, providing a reference for the clinical evaluation of treatment efficacy and prognosis of patients.

Functional activation of a novel R2R3-MYB protein gene, GmMYB68, confers salt-alkali resistance in soybean (Glycine max L.).

Soil salinity significantly reduces soybean (Glycine max L.) production worldwide. Plants resistance to stress conditions is a complex characteristic regulated by multiple signaling pathways. The v-Myb avian myeloblastosis viral oncogene homolog (MYB) transcription factor (TF) plays a crucial role in plant development, secondary metabolism, and abiotic stress responses. GmMYB68-overexpression and RNA interference (RNAi) lines were established for examining the function of G. max GmMYB68 in plant responses to abiotic stresses. The predicted amino acid sequence of GmMYB68 was similar to that of R2R3-MYB proteins. Quantitative real-time PCR analysis revealed that GmMYB68 expression varied in response to abiotic stresses. GmMYB68-overexpression lines showed enhanced resistance to salt and alkali stresses and their osmotic adjustment and photosynthetic rates were also stronger than that of GmMYB68-RNAi and wild type plants. Although wild type and transgenic plants showed no significant differences in agronomic traits under normal conditions, the overexpression of GmMYB68 increased grain number and 100-grain weights under salt stress. Our study identified a valuable TF associated with stress response in soybean, as its overexpression might help improve salt and alkali tolerance in soybean and other crops.

Discovery of the Agrobacterium growth inhibition sequence in virus and its application to recombinant clone screening.

Infectious clone vectors used widely in genetic research. While constructing soybean mosaic virus (SMV) clone vectors, we found that transformed Agrobacterium grew significantly different depending on the viral strains used. In particular, the clone vectors constructed with SMV SC15 significantly suppressed the growth of Agrobacterium. Recombinant and truncated virus vector experiments showed that the polymorphism of a P1 protein coding sequence of SC15 leads to the growth inhibition of Agrobacterium. But the lack of other protein encoding sequences, except for the sequence encoding coat protein, should reduce the ability of SC15 to suppress Agrobacterium growth. A vector (pCB301-attL-SC15P) compatible with the Gateway cloning system was constructed using this Agrobacterium inhibitory sequence. The results from the LR recombination reaction with pCB301-attL-SC15P and Agrobacterium transformation showed the valuable application potential of the Agrobacterium inhibitory sequence to serve as a negative screening factor for effective recombinant clone screening in Agrobacterium.

Introduction of the harpinXooc-encoding gene hrf2 in soybean enhances resistance against the oomycete pathogen Phytophthora sojae.

Phytophthora root and stem rot (PRR) caused by an oomycete pathogen Phytophthora sojae is one of the most devastating and widespread diseases throughout soybean-producing regions worldwide. The diversity and variability of P. sojae races make effective control of the pathogen challenging. Here, we introduced an elicitor of plant defense response, the harpinXooc-encoding hrf2 gene from the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola into soybean and evaluated resistance to P. sojae infection. Molecular analysis confirmed the integration and expression of hrf2 in the transgenic soybean. After inoculation with P. sojae, non-transformed control (NC) plants exhibited typical PRR symptoms, including necrotic and wilting leaves, and plant death, whereas most of the transgenic plants showed slightly chlorotic leaves and developed normally. Through T3 to T5 generations, the transgenic events displayed milder disease symptoms and had higher survival rates compared to NC plants, indicating enhanced and stable resistance to P. sojae infection, whereas without P. sojae inoculation, no significant differences in agronomic traits were observed between the transgenic and non-transformed plants. Moreover, after inoculation with P. sojae, significant upregulation of a set of plant defense-related genes, including salicylic acid- and jasmonic acid-dependent and hypersensitive response-related genes was observed in the transgenic plants. Our results indicate that hrf2 expression in transgenic soybean significantly enhanced resistance to P. sojae by eliciting multiple defense responses mediated by different signaling pathways. The potential functional role of the hrf2 gene in plant defense against P. sojae and other pathogens makes it a promising tool for broadening disease resistance in soybean.

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1.

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase.

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T3 and T4 generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T4 generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H2O2 at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.

Overexpression of a novel transcriptional repressor GmMYB3a negatively regulates salt-alkali tolerance and stress-related genes in soybean.

Myeloblastosis (MYB) transcription factor (TF) plays a positive role in the growth and stress response of plants; however, information on the functions of MYB repressors in soybean is limited. In the present study, the gene GmMYB3a was identified and characterized as a member of the R2R3 MYB repressor family, which is induced by various abiotic stresses. To understand the functions of GmMYB3a, a transgenic soybean over-expressing GmMYB3a was obtained and the photosynthetic index under salt-alkali treatments was evaluated. The transgenic line exhibited a series of negative regulation relative to the wild-type control. Quantitative real time polymerase chain reaction revealed that the physiological parameters, including soluble sugar, free proline, and chlorophyll contents; and photosynthetic rate decreased in the transgenic plants. Furthermore, GmMYB3a overexpression down-regulated a set of key genes associated with plant defense signal pathways. These finding suggest that GmMYB3a negatively affects the response of plants to salt stress.

Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B.

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the ß-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~ 80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total protein and oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.

RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean.

KEY MESSAGE: Robust RNAi-mediated resistance to multiple Potyvirus strains and isolates, but not to Secovirus BPMV, was conferred by expressing a short SMV P3 hairpin in soybean plants. Engineering resistance to multiple Potyvirus strains is of great interest because of a wide variability of the virus strains, and mixed infections of multiple viruses or strains commonly associated with field grown soybean. In this study, RNAi-mediated silencing of the soybean mosaic virus (SMV) P3 cistron, which is reported to participate in virus movements and pathogenesis and to be the putative determinant of SMV virulence, was used to induce resistance to multiple Potyvirus strains and isolates in soybean. A 302 bp inverted repeat (IR) of the P3 cistron, isolated from the SMV strain SC3, was introduced into soybean. The transgenic lines exhibited stable and enhanced resistance to SMV SC3 under field conditions over 3 consecutive years. The transgenic lines also showed significantly enhanced resistance to four other SMV strains (SC7, SC15, SC18, and SMV-R, a novel recombinant found in China), the soybean-infecting bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV). Nevertheless, no significant differences were found between transgenic plants and their non-transformed (NT) counterparts in terms of resistance to bean pod mottle virus (BPMV, Secoviridae). Consistent with the results of resistance evaluations, the expression of the respective viral CP cistrons and virus accumulation were significantly lower in seven Potyvirus strains and isolates than in the NT plants, but not in BCMV-inoculated transgenic lines. The results demonstrate the effectiveness of engineering resistance to multiple Potyvirus strains and isolates via RNAi-mediated SMV P3 cistron silencing, and thus provide an effective control strategy against Potyvirus infections in soybean and other crops.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

[Fast Identification of Transgenic Soybean Varieties Based Near Infrared Hyperspectral Imaging Technology].

Near-infrared hyperspectral imaging technology combined with chemometrics was applied for rapid and non-invasive transgenic soybeans variety identification. Three different non-GMO parent soybeans(HC6, JACK, TL1)and their transgenic soybeans were chosen as the research object. The developed hyperspectral imaging system was used to acquire the hyperspectral images in the spectral range of 874~1 734 nm with 256 bands of soybeans, and the reflectance spectra were extracted from the region of interest (ROI) in the images. After eliminating the obvious noises, the moving average(MA)was applied as smooth pretreatment, and the wavelengths from 941~1 646 nm were used for later analysis. Partial least squares-discriminant analysis (PLS-DA)was employed as pattern recognition method to class the three different non-GMO parent soybeans. The classification accuracy of both the calibration set and the prediction set were 97.50% and 100% for the HC6, 100% and 100% for the JACK, 96.25% and 92.50% for the TL1, which indicated that hyperspectral imaging technology could identify the varieties of the non-GMO parent soybeans. Then PLS-DA was applied to classify non-GMO parent soybean and its transgenic soybean cultivars for building discriminant models. For the full spectra, the classification accuracy of both the calibration set and the prediction set were 99.17% and 99.17% for the HC6 and its transgenic soybean cultivars, 87.19% and 81.25% for the JACK and its transgenic soybean cultivars, 99.17% and 98.33% for the TL1 and its transgenic soybean cultivars, respectively. The sensitive wavelengths were selected by x-loading weights, and the classification accuracy of the calibration set and prediction set of PLS-DA models based on sensitive wavelengths were 72.50% and 80% for the HC6 and its transgenic soybean cultivars, 80.63% and 79.38% for the JACK and its transgenic soybean cultivars, 85% and 85% for the TL1 and its transgenic soybean cultivars, respectively. These results showed that the pattern recognition for non-GMO parent soybean and their transgenic soybeans was feasible, and the selected sensitive wavelengths could be used for the pattern recognition of non-GMO parent soybeans and transgenic soybeans. The overall results indicated that it was feasible to use near-infrared hyperspectral imaging technology for the pattern recognition of the non-GMO parent soybeans varieties, non-GMO parent soybean and its transgenic soybeans. This study also provided a new alternative for rapid and non-destructive accurate identification of transgenic soybean.

Changes in oleic Acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.

The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

Isolation of a novel C18-Δ9 polyunsaturated fatty acid specific elongase gene from DHA-producing Isochrysis galbana H29 and its use for the reconstitution of the alternative Δ8 pathway in Saccharomyces cerevisiae.

A novel gene (IgASE2) encoding a C18-Δ9 polyunsaturated fatty acids specific (C18-Δ9-PUFAs-specific) elongase was isolated and characterized from DHA-rich microalga, Isochrysis galbana H29. The IgASE2 gene was 1,653 bp in length, contained a 786 bp ORF encoding a protein of 261 amino acids that shared 87% identity with Δ9 elongase, IgASE1, and possessed a 44 bp 5'-untranslated region (5'-UTR) and a 823 bp 3'-untranslated region (3'-UTR). IgASE2, by its heterologous expression in Saccharomyces cerevisiae, elongated linoleic acid (LA, 18:2n-6) and α-linolenic (ALA, 18:3n-3) to eicosadienoic acid (EDA, 20:2n-6) and eicosatrienoic acid (ETrA, 20:3n-3), respectively. The conversions of LA to EDA and ALA to ETrA were 57.6 and 56.1%, respectively. Co-expression of this elongase with Δ8 desaturase required for the synthesis of C20-polyunsaturated fatty acids resulted in the accumulation of dihomo-γ-linolenic acid (20:3n-6) from LA and eicosatetraenoic acid (20:4n-6) from ALA. These results demonstrated that IgASE2 exhibited C18-Δ9-PUFAs-specific elongase activity and the alternative Δ8 pathway was reconstituted.

[Cloning of delta8-fatty acid desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae].

Delta8 desaturase pathway, different from common delta6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Delta8-fatty acid desaturase is one of the key enzymes in delta8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic acid and omega3-eicosatrienoic acid for the expression of delta8-fatty acid desaturase gene. The results indicated that high level expressed As-fatty acid desaturase could convert omega6-eicosadienoic acid and omega3-eicosatrienoic acid to dihomo-gamma-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.